Fermentation process



Patented July 12, 1949 FERMENTATION PROCESS Leonard B. Schweiger andRaymond L. Snell, Elkhart, Ind., assignors to Miles Laboratories, Inc.,Elkhart, Ind., a corporation of Indiana No Drawing. Application July 16,1948, Serial No. 39,173

9 Claims.

This invention relates, generally, to improvements in the production ofcitric acid by oxidative, submerged fermentation of carbohydrates underthe action of a citric acid producing strain of Aspergillus niger. Moreparticularly, the invention relates to such a method of producing citricacid wherein both ammonium carbonate and morpholine are present in thebasal medium.

In our previous application, Serial No. 741,108, filed April 12, 1947,to which reference is hereby made, we have disclosed a method, andsuitable apparatus, for producing citric acid by oxidative,

submerged fermentation of carbohydrate material under the action of A.niger using ammonium carbonate as the source of nutrient nitrogen. Wehave now found that our previous process may be materially improved,particularly with respect to increased yields of citric acid, by havingboth ammonium carbonate and morpholine present in the media undergoingfermentation.

The object of the invention, generally stated, is the provision of animproved method of producing citric acid by oxidative, submergedfermentation of assimilable carbohydrate materials An aqueous solutionof the following composition was prepared:

Composition A Ammonium carbonate per cent by weight 0.2 KH2PO4 do 0.014MgSO4.7H2O do 0.1 Refined sugar do 13.1 Decationized well water, 41. s.4 liters 500 P. P. M. (.05%) of morpholine were added to Composition Aand its pH value was adjusted to approximately 2.5 by addition ofconcentrated hydrochloric acid, and it was then introduced into a columntype fermenter in the form of a Pyrex column having an inside diameterof three inches and a length of forty-eight inches. This type ofapparatus is shown in Figure 2 of the drawings forming a part of saidapplication, Serial No. 741,108. As a precaution, the fermentingapparatus before use was previously sterilized with steam for a periodof 2-3 hours. The medium was sterilized in a Pyrex container by placingthe container in an autoclave and introducing steam at 10 pounds persquare inch. After a period of about twenty minutes the container andits contents were cooled to approximately room temperature.

The sterilized medium was next introduced into the sterile fermenter andinoculated with a spore suspension of a strain of A. niger identified as18B2 and characterized by its good acid producing ability. The sporesuspension was prepared by washing three to six day old growth from agarslants with sterile distilled water. Suflicient spore suspension wasused to give a spore concentration in the basal medium of approximately50-100 million spores per liter, as determined by the hemocytometer orLumatron methods.

Sterile humidified air at a temperature of about 35 C. was passedthrough the column at a rate of approximately 4 liters per minute andthe fermentation process was continued for nine and two-thirds days withthe temperature of the medium being maintained at 30-32 C.

At the end of this time, fermentation was discontinued and the contentsof the fermenter were withdrawn and the citric acid recovered therefromby precipitation in the form of calcium citrate, in accordance withknown procedure. In terms of citric acid produced, the fermentationresulted in the production of 384 grams of citric acid. Based on theinitial amount of refined sugar in the medium, this amount of citricacid represents a 77.1% conversion of the initial sugar to the acid.Approximately 30 grams of unconsumed sugar remained in the fermentedmedium, so that based on the amount of sugar consumed, 94% thereof wasconverted to citric acid. No detectable amount of oxalic acid wasproduced during the fermentation.

EXAMPLE II The following medium for fermentation was prepared using awell water decationized by passage through a cation exchange resinoperating on the hydrogen cycle, such, for example, as the phenolformaldehyde type cation exchange resin sold under the trade nameAmberlite IR-l:

Composition B Ammonium carbonate per cent by weight 0.2 KH2PO4 do 0.014MgSO4.7H2O do 0.1 Refined sugar do 12.0 Decationized well water, q. s .4liters 1000 P. P. M. (.1%) of morpholine were added to Composition B andit was fermented in the apparatus, and in accordance with the proceduredescribed above in connection with Example I," except that thefermentation was allowed to run only eight and two-thirds days. Thequantity of citric acid produced was 309 grams, which amount constitutesa 61.8% conversion of the initial sugar present in the medium and a93.1% conversion of the sugar consumed.

. Exmrtr: III The following medium for fermentation was prepared byknown decationization techniques:

Composition C Ammonium carbonate percent by weight 0.2 KHzPO4 do 0.014

' MgSO4..7HzO do 0.1

Decationized raw sugar do 12 8 Decationized well water, q. s .4 liters500 P. P. M. (0.05%) morpholine were added to Composition C and it wasthen fermented in the apparatus, and in accordance with the proceduredescribed under Example I, except that the fermentation was discontinuedafter 8% days. 79.9% of the initial sugar was converted to citric acid.

EXAMPLE IV The fermentation of Example III was repeated using 13.5%decationized corn syrup instead of the decationized raw sugar. The cornsyrup was decationized in one pass and then diluted to contain 13.5%fermentable carbohydrate. 61.5% of the initial fermentable material wasconverted to citric acid.

The results of Examples III and IV show that such relatively inexpensiveash-containing raw materials as raw sugar and corn syrup, can 59.5151

factorily be substituted for refined sugar as substrates for citric acidproduction, especially if such raw materials are first suitablyconditioned for this purpose by decationization.

EXAMPLE V In order to demonstrate that the excellent yields ofcitricacid obtained in the relatively small scale fermentations of theprevious examples,

were reproducible on a commercial scale, termentations in accordancewith the present invention were conducted in a pilot plant using acolumn type fermenter approximately 24 feet high with a diameter ofabout 19 inches. In one such fer-j mentation 275 gallons of mediumhaving the following composition were fermented:

Percent by weight Ammonium carbonate U. S. P. 0.2 KH2PO4 0.014LTgSOn'TI-IzO U. S. P. 0.1 Morpholine 0.05 i Refined sugar 13.0

Zn++, .1-.4 P. M. pH=2.5 2.6 with HCl.

At the end of 8% days of fermentation, the medium was harvested,yielding the following data:

Media volume gal. 275

Initial sugar, wt. lbs. 298.5 Residual sugar, conc percent 3.75 Residualsugar, wt lbs 86.2 Titrable acidity, as citric percent 8.61 Oxalic acidNegative Total acid, wt lbs 198 Sugar consumed lbs. 2123 Percentconversion of original sugar 66.5%

(yield) Percent conversion of consumed sugar 93.5%

(efficiency) In each of the fermentations carried out under theforegoing examples, the cellular morphology was so controlled as toinduce cell structure characterized by: i

a. Abnormally short, stubby, forked, bulbous mycelium.

b. Numerous swollen, oval to spherical-shaped cells well distributedthroughout the mycelial structure.

0. Mycelial structures all showing granulations, and numerous vacuolesor refractile bodies.

d. Absence of normal reproductive bodies (vesicles or sterigmata).

e. Formation of compact aggregates or colonies having a gross granularappearance and of sizes under 0.5 mm. in cross section and averagingabout 0.1 mm.

The photomicrographs shown in Figures 3, 4 and 5 of said application,Serial No. 741,108, show cell structure having the abovecharacteristics.

The mycelium in each instance was non-slimy, and of a granular naturepermitting ready separation from the substrate, by filtration.

The three following tables present data showing the effect of varyingconcentrations of morpholine on citric acid production:

TABLE I Effect of varying concentrations of morpholine on citric acidproduction (using A. Niger 1882) Pig cent Siuear Cone. of onYers on 133}3 Morpholine, g

P. P. M. Com

Initial I sumed A. Composition A... 0. 0 313.0 62.4 87. 7 B. CompositionA..- 10. 0. 001% 324.0 64. 5 87. 2 C. Composition A--- 50. 0. 005% 307.0 61. 2 87. 2 D. Composition A... 100. 0-. 01% 375.0 75. 3 89.9 E.Composition A..- 500. 0-. 05% 384. 0- 77. 1 94. 0

g'lotal acidity in 9% days fermentation.

TABLE II Efie ct of varying concentrations of morpholine on citric acidproduction (using A. Niger 18132) Per cent Sugar, Basal Media Acidity iconversion Per cent Morpholmm g.

P. P. M. Com

Innis sumed A. Composition B 0.0 294. 0 58.8 83.3 B. Composition B--.500.0 334.0 06.8 93.8 C. Composition B--- 1000. 0 300. 0 61. 8 93. 1

. Total acidity in 8% days fermentation.

TABLE III Eflect of morpholine on citric acid production (using A. niger1882) Per cent Bug. Cone. of Conv. P Basal lgdiat, t MgrpholAcid...Residual erccn' weg ne, 'tyJg. Su'ar P. P.M. Ini- Contial sumed A.Ammonium Carbonate, 2 KHzPOr, 0.014"-.- MgBO4.7Ha0, 0.1.. 0.0 365.0 74.088.0 78 Sucrose, 12.8 Decationized well Water, q. s.4 l- B. Same as A500.0 389.0 80.4 05.6 77

1 Total acidity in 9% days fermentation.

From the foregoing data, it appears that the morpholine concentrationmay effectively range from 100 to 1000 P. P. M., with 500 P. -P. M.being an approximate optimum concentration.

Table IV below contains data showin that the improved effect ofmorpholine is not restricted to a single strain of A. nicer. Strain No.139 was obtained by ultraviolet irradiation of spore suspensions ofstrain 18B2. The spores of strain No. 139 are tan, whereas the spores ofthe current 1832 are jet black.

TABLE IV Efiect of morpholine on citric acid' productionby Aspergillusniger 18Bz'cnd Aspergillus niger 139 f Pg!" cent Cone. of Basal MediaConvers.

Organ- Morpho- Acid- Per cent by weight ism line 8' P. P. M. hm Com tialsumed A. NH CO 0.2 18B; 500. 0 347.0 70.5 98.0 KHzPO4, 0.014"-.. 18131 00. 255. 0 51. 82. 5 MgSO4, 0.1 Refined sugar, 12.7. 139 500.0 326. 0 66.5 97. 3 Well Water, q. s.-

1 Total acidity in 7% days fermentation.

Efiect of various concentrations of morpholine and ammonium carbonate oncitric acid yields Percent Sugar Conversion (NH4)2C 0;, Morpholine,Acidity,

Percent Percent g. C

on- Initial sumed a 0. 20 0. 00 190 38. 0 84. l b 0. 20 0. 05 246 49. 296. 1 c 0. 05 0. 95 19. 0 89. 6 d 0. 00 0. 0 0. 0 0. 0 e 0. 00 0. l0 00. 0 0. 0

ammonium carbonate proximately 0.2-0.15

In fermentations d and e, having morpholine as the sole source ofnitrogen, mycelial growth was scanty, abnormal in cellular morphology(when. compared with the cell structure shown in Figures 3. 4 and 5 ofsaid application, Serial No. 741,108, as normal), and produced no citricacid. When morpholine was used as the major source of nutrient nitrogen,but was supplemented with 0.05% (viz. fermentation 0), growth was stillabnormal but some acidity was produced. Ammonium carbonate alone (viz.fermentation a) produced a good yield of acid for the fermentationperiod involved, but the yield was enhanced greatly when 0.2% ammoniumcarbonate was used as the major nitrogen source supplemented with 0.05%morpholine (viz. fermentation b). It is apparent from the data containedin Table V that morpholine and ammonium carbonate, in properconcentrations,

produce some associated effect on the yield of citric acid by A. niger.

As pointed out above, the morpholine may range in concentration from 100to 1000 P. P. M. with approximately 500 P. P. M. being optimum.Experience has shown that the ammonium carbonate concentration may rangebetween ap- (The chemical which is commonly known as ammonium carbonateis actually a mixture of ammonium bicarbonate and ammonium carbamate andcontains approximately 30-34% by weight of ammonia. and approximately45% C02.) The KH2PO4 may range from 0.01 to 0.02% and the MgSO4.7HzO mayrange from 0.08 to 0.15% by weight. The fermentable carbohydrate orsugar material may range in concentration from 10 to 15% by weight, withfrom 12 to- 13% bein the preferred range. Refined sugar may be used, orlow-ash containing sugar materials, such as raw sugar, corn syrup orhigh-test molasses may be used. These latter materials, for goodresults, must be decationized so as to reduce the Fe concentration tonot more than about 1 P. P. M. The pH value of the basal media may rangebetween 2.5 and 2.6. No more than one part per million of Fe should bepresent, and if it is present, the Zn++ should range in concentrationbetween .00005% 001%, so as to counteract the efiect of the Fe on thecell morphology.

The above data shows that morpholine in th presence of ammoniumcarbonate has the following important effects on citric acid productionin the submerged state:

1. Increases the conversion of initial carbohydrate, with as high asconversions being obtained in practical fermentation times of 8-10 days.

2. Produces increased efliciency of fermentation, that is, of thesubtrate consumed, a higher precent is converted to citric acid.

3. Stimulates early germination of spores, resulting in a faster rate ofacid production in th early stages of fermentation.

4. Contributes a stabilizing effect on the physiological characteristicsof the fungus, permitting better duplication of results.

At the present time, we are not able completely to elaborate the roleplayed by the morpholine. It is postulated that the morpholine may serveas a supplementary nitrogen source which requires more energy for itsutilization, thus placing a greater requirement on the physiologicalmechanism of the fungus.

The fermentations may be carried out in tank and avenue 7 g typefermenters. such as the one shown in Figure 1 of said application SerialNo. 741,108. In fact, the particular type and construction of thefermenters which may be used are not critical.

Sterile, humidified airis the preferred aerating medium. However, ifeconomically feasible, other oxygen-containing gases may be used.Foaming in the fermenters may be controlled by addition to the contentsof suitable antifoam agents, such as, octadecyl alcohol in mineral oilor Tween 81.

Numerous modified techniques may be adopted in carrying outfermentations according to our invention. Thus, additional amounts ofthe sugar material may be added during the course of the fermentations.Instead ofadding the spore suspensions directly to the basal media,mycelial mats may first be grown in a similar media and then maceratedand added to the contents of a fermenter. Likewise, preformed submergedcells may be developed and used with a view toward reducing oreliminating the incubation period for the spores in the fermenters. 1

In view of the foregoing disclosure, those skilled in the art will beable advantageously to practice the invention, as well as make certainfurther modifications in the specific embodiments described above.yention is to be limited only within the limits defined in the appendedclaims. 1

We claim: I

1. In the production of citric acid by sub I merged oxidativefermentation of assimilable carbohydrate-containing. media with a citricacid producing strain of A. niger, the improvement which comprisesincorporating ammonium carbonate and morpholine in the media. I

2. A method of producing citric acid by s. merged fermentation, whichcomprises, subjecting a sugar material to the action of a citric acidproducing strain of A. niger in an agitated, aqueous solution containingin addition to said sugar material nutrient mineral substances andammonium carbonate and morpholine, and passing an oxygen-containing gasthrough said solution.

3. A method of producing. citric acid by sub merged fermentation, whichcomprises, subjecting a sugar material to the action of a citric acidproducing strain of A. niger in an agitated, aqueous solution containingin addition to said sugar material nutrient mineral substances and, fromabout 0.1 to 0.3% by weight of ammonium carbonate, and from 100-1000 P.P. M. of morpholine,

and passing an oxygen-containing gas through said solution.

4. The process of claim 3 wherein said oxygen-j containing gas is air.

5. A method of producing citric acid by submerged fermentation, whichcomprises, subjecting a sugar material to the action of a citric acidproducing strain of A. niger in an agitated aqueous solution of saidsugar material having a pH of about 2.5-2.6 and containing from about0.1

Accordingly, the scope of the in- 8 to 0.3% by weight of ammoniumcarbonate and from about 100-l000 P. P. M. of morpholine as well asnutrient concentrations of KH2PO4 and MgSO4.7I-I:O, and passing anoxygen-containing gas in finely dispersed condition through saidsolution.

6. The method called for in claim 5 wherein said KHzP04 phosphate has aconcentration from about 0.01% to 0.02% and said MgSO4.7H2O has aconcentration from about .08 to .15%.

7. A method of producing citric acid by submerged fermentation, whichcomprises, subjecting a sugar material to the action of a citric acidproducing strain of A. niger in an agitated aqueous solution having a pHof about 2.5-2.6 and containing about 12% by weight of refined sucrose,about 0.2% by weight of ammonium carbonate, about 500 P. P. M. ofmorpholine, about 0.014% by weight of KH2PO4 and about 0.1% ofMgSO4.7H2O, and passing air in finely dispersed condition through saidsolution.

8. A method of producing citric acid by submerged fermentation whichcomprises subjecting a sugar material to the action of a citric acidproducing strain of A. niger yielding a cell structure characterized by:

x a. Abnormally' short, stubby, forked, bulbous in an agitated, aqueoussolution of said sugar material having a pH of about 2.5-2.6 andcontaining from about 0.1-0.3% by weight of ammonium carbonate and fromabout 500-4000 P. P. M. of morpholine, as well as nutrientconcentrations of KH2PO4 and MgSO4.7H2O, and passing air in finelydispersed condition through said solution.

9. A method of producing citric acid by submerged fermentation, whichcomprises, subjecting an ash-containing sugar material decationized toreduce the Fe content to not more than 1 P. P. M. to the action of acitric acid producing strain of A niger in an agitated aqueous solutioncontaining in addition to said decationized sugar material nutrientmineral substances includin from about .00005 to .001% Zn to counteractsaid Fe content, and ammonium carbonate and morpholine, and passing anoxygen-containing gas through said solution.

LEONARD B. SCHWEIGER.

RAYMOND L. SNELL.

No references cited.

